6 Department Circular 28b, U. S. Dept. of Agriculture 



piece was then inclosed in a piece of filter paper to protect it from 

 dust and was allowed to dry at the ordinary room temperature of 

 the laboratory until no odor of formalin could be detected. 



CULTURES 



Yeast-extract egg-yolk agar medium, as described in a recent paper 

 by the writer (#i, p. 136) was used in the form of slants for the pur- 

 pose of making the cultural tests of the scales treated by the various 

 solutions. In order that there should be a sufficient amount of water 

 of condensation at the base of the slants, the necessary number of 

 tubes of fresh medium for each group of combs to be tested were 

 made up by the addition of sterile egg yolk to tubes of the base 

 medium. With each new lot of medium control cultures were made 

 from untreated scales of American foulbrood. 



The removal of scales or other diseased remains from the cells of 

 the treated combs was accomplished by means of a lancet-shaped 

 dissecting needle which had just been sterilized in a flame and 

 allowed to cool. The scale (or other remains) was then carefully 

 placed in the water of condensation of the tube of culture medium, 

 one scale or material from one cell to a tube of medium. Cappings 

 from sealed brood were removed with the hot needle, and after 

 resterilizing the needle the scale contained in one cell was removed 

 and placed in a culture tube. After the diseased material had been 

 allowed to soak in the condensation water of the tube for about an 

 hour, or longer if necessary, until the dried scale had softened, it 

 was macerated and spread over the surface of the slant by means of 

 a stiff platinum loop. 



Cultures were at first made of material from one open cell and one 

 sealed cell from each comb treated, on the assumption that for a 

 given solution the sterilization would be uniform for a definite period 

 of time, but it soon became evident that there was a variation in the 

 rapidity with which the various solutions penetrated the brood cap- 

 pings. At least five cultures were therefore made from open cells 

 and five from sealed cells in each piece of comb, amounting to ap- 

 proximately 3 to 5 per cent of all cells in a piece of comb of the size 

 used in these experiments. The percentage cultured of the scales 

 actually present is really much higher than this, since a piece of 

 comb of that size seldom has a scale in every cell. 



After incubation for 48 hours at 37° C, cover-glass smears were 

 made from the material on the surface of the slants. A large loop- 

 ful of a mixture of material from different parts of the slant in a 

 drop of distilled water was used in making a good-sized smear on 

 the cover glass. After drying the cover glass in the air and passing 

 it quickly through a flame three times the smears were stained for 

 about 20 seconds with Ziehl-Neelson carbol-fuchsin diluted with an 

 equal quantity of distilled water. The cover glasses were then care- 

 fully washed in water, dried, mounted with Canada balsam, and 

 examined under the microscope. At least 20 to 30 fields were ex- 

 amined to determine whether there had been any germination of 

 spores of Bacillm larvae or vegetative growth not visible on the 

 slant. When no germination of spores was observed, as a rule only 

 one examination of the culture was made at the end of 48 hours' 

 incubation, since if there are any spores in a condition to germinate 



